Automated assay machine and assay tray

ABSTRACT

A machine for transferring liquids to and from the wells of assay trays in a controlled, automated manner and a solid phase assay tray for use with the machine. The machine includes a horizontally translatable table (15) that holds the tray (46), a plurality of liquid dispensing manifolds (54) for dispensing liquids into the tray wells (50) and an aspirating manifold (65) for aspirating liquid from the well. The dispensing and aspirating manifolds are mounted on a vertically translatable head (16) above the table. Each dispensing manifold is equipped with a row of dispensing tubes (56) and is connected via a pump (58) to a liquid container (62). The aspirating manifold is equipped with a row of aspirating tubes (66) and is connected via a pump (69) to a waste liquid receptacle (73). A microprocessor (85) controls the movements of the table and manifolds and operates the pumps. The tray wells include means, such as sloping bottoms (74) or subwells (83), that cause the solid phase (76) to occupy a particular position in the wells and sumps (75), (84) that are positioned relative to the location of the solid phase such that they may be accessed vertically by the aspirating tubes without danger of disturbing the solid phase. The bottoms of the sumps have optically flat areas so that beams of light may be passed vertically through the liquid contents of the wells without intersecting the solid phase to make optical measurements of the liquid contents.

This application is a division of application Ser. No. 889,797, filedJuly 24, 1986 abandoned, which is a division of application Ser. No.656,234, filed Oct. 1, 1984 now U.S. Pat. No. 4,681,742.

DESCRIPTION

1. Technical Field

This invention is in the field of immunoassay apparatus. Moreparticularly it concerns (a) an immunoassay tray for carrying out animmunoassay in which an immunochemical is fixed to a solid phase and theimmunochemical-bearing solid phase is incubated with liquid reagents andwashed with wash liquids and (b) a machine for use in combination withthe tray for depositing and withdrawing the reagent/wash liquids fromthe tray wells.

2. Background Art

Immunoassays are used to detect the presence or quantity of a givenimmunochemical in a sample suspected of containing the immunochemical.Most immunoassays involve the formation of immune complexes viaantigenantibody binding. The complexes are detected in various ways,such as by labels bound directly or indirectly to the complex.

Many immunoassays require that the complexes be subjected to one or moreisolations, washings, and treatments with liquid reagents. In order tofacilitate handling the immune complexes, one of the components of thecomplex is used in a solid phase form (i.e., immobilized on aparticulate immunosorbent material) which results in the complexes alsobeing in solid phase. The immobilized complexes may be easily washed,incubated with additional reagents, and isolated as required by theparticular type of assay. Assays that employ a particulate solid phasereagent are commonly referred to as "solid phase", "heterogeneous", or"immunosorbent" assays. For convenience they will be referred to hereinas "solid phase" immunoassays.

Solid phase assays are frequently carried out in assay trays or tubes.Assay trays are plates that have a plurality of wells (usually 20, 24,48 or 96) arranged in rows and columns into which the particulate solidphase is placed and treated sequentially with the liquid reagents andwashes involved in the particular assay. Various well configurationshave been used or suggested, including wells having flat bottoms,V-shaped bottoms, and U-shaped bottoms. The liquid reagents and washesare normally added and withdrawn from the wells with manual,semiautomated or automated pipettes.

With most of the current assay trays the location of the solid phase inthe well bottom is happenstance. Accordingly, when the pipette tip isinserted into the well there is a possibility of hitting the solid phaseand mechanically dislodging bound materials from its surface. Also, thesolid phase may interfere with withdrawing liquids completely from thewell. In view of this, many assay protocols require that the solid phasebe removed from the wells prior to liquid transfer and replaced in thewells after liquid transfer. Also in some types of assays, such asfluorescent immunoassays (FIAs) and enzyme immunoassays (EIAs), thereading step of the assay involves passing a beam of light through theliquid phase. If the solid phase is positioned randomly in the wells itmay interrupt the path of the beam, requiring adjustment of the beampath or removal of the solid phase from the well.

Manual, semiautomated or automated pipettes are commonly used for theliquid transfer steps of immunoassays. An example of a fully automatedpipette system that may be used to carry out an immunoassay is describedin commonly owned U.S. application Ser. No. 06/489,866, filed May 5,1983. The extensive number of steps and reagents involved in manyimmunoassays may, however, make it impractical to use such systems wherethe reagents are carried in open receptacles on a moving table and aretransferred via pipette. Also, these pipette systems are generally notcapable of carrying out the reading phase of an immunoassay. Theinvestigator must, therefore, transfer the assay tray to readingapparatus. In sum, current assay equipment requires substantial humanintervention and is not capable of conducting assays fromstart-to-finish automatically.

Among the objects of the present invention are to provide a novel assaytray whose wells include means for positioning the solid phase such thatit does not interfere with the implement(s) used to introduce andwithdraw liquid from the wells or optical measurements made on theliquid contents of the well at the end of the assay and a machine thatintroduces and withdraws liquids from such wells in an automatic,controlled fashion without necessarily using pipettes and can be adaptedto carry out assays automatically from start-to-finish.

Use of the trays of the invention avoids handling of the solid phase,improves the reliability of the assay, and permits solid phase assays tobe conducted automatically. The invention machine is particularlyadapted to be used in combination with the trays and provides a meansfor carrying out solid phase assays from start-to-finish with no or onlyminimal operator monitoring and intervention. In this regard the machinemay be readily equipped to conduct the reading phase of the assayautomatically and to communicate with a microprocessor to record assaydata, correlate such data with other data (if necessary or desired),process or analyze the data, and prepare reports based on the data. Whenequipped with assay reading means and linked to a microprocessorprogrammed with suitable software, the present invention provides aself-contained system for conducting, recording, and reporting assays.

STATEMENT OF THE INVENTION

There are two principal aspects of the invention: (1) novel assay traysfor use in solid phase assays and (2) a machine for supplying andwithdrawing liquid reagents to and from the wells of assay trays in anautomated controlled manner. These aspects are broadly described below.

The assay trays are used in assays wherein a a particulate solid phase,typically carrying an immobilized reagent, is contacted with one or moreliquids. They include means for positioning the solid phase at apredetermined location in the wells combined with other novel structuralfeatures that permit liquid to be aspirated from the wells withoutdisturbing the solid phase or the solid phase to be immersed in aminimal volume of liquid or facilitate automated reading of the liquidin the wells.

Thus, in one embodiment the invention tray comprises a plate having aplurality of wells for receiving the solid phase and liquids, the wellshaving:

(a) a solid phase positioning means for positioning the solid phase in apredetermined location in the wells; and

(b) a sump means spaced from the location of the solid phase wherebyliquid may be withdrawn from the wells without disturbing the solidphase.

In another embodiment the invention tray comprises a plate having aplurality of wells for receiving the solid phase and the liquids, thewells each having:

(a) a subwell section for holding the solid phase, the size and shape ofthe subwell section being such that the solid phase may be immersed inthe subwell section in a small volume of liquid; and

(b) a main well section of substantially greater cross sectional areaand volume than the subwell section.

The machine comprises in combination:

(a) a head assembly translatable between upper and lower positions alonga vertical axis and including:

(i) at least one liquid reagent dispensing manifold having an inletadapted to be connected to one or more liquid reagent sources and aplurality of spaced outlets each fitted with a vertically dependingliquid reagent dispensing tube, the spacing between the dispensing tubesbeing in correspondence with the spacing between the wells of the assaytray; and

(ii) at least one liquid reagent aspirating manifold having an outletand a plurality of spaced inlets each fitted with a vertically dependingliquid reagent aspirating tube, the spacing between the aspirating tubesbeing in correspondence with the spacing between the wells of the assaytray;

(b) dispensing pump means operably associated with the source(s) ofliquid reagent and the dispensing manifold inlet(s) for pumping liquidreagent from the source(s) into the dispensing manifold(s) wherebyliquid reagent is deposited in the wells;

(c) aspirating pump means operably associated with the outlet(s) of theaspirating manifold(s) for creating a vacuum therein whereby liquidreagent is aspirated from the wells;

(d) means for moving the head assembly along its vertical axis;

(e) a table mounted beneath the head and being adapted to carry theassay tray, the table and/or head being moveable horizontally relativeto one another;

(f) means for moving the table and/or head horizontally to position therows of wells in registry with the dispensing tubes and aspiratingtubes; and

(g) means for controlling each of the moving means for the head andtable, the dispensing pump means, and the aspirating pump means wherebypredetermined volumes of liquid reagents are deposited and aspiratedfrom the wells in a predetermined sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 is a perspective, partly broken-away view of a prototypeembodiment of the assay machine of the invention;

FIG. 2 is a partly sectional, partly broken-away side elevational viewof the machine of FIG. 1, showing the head assembly of the machine inits upper position;

FIG. 3 is an enlarged sectional view taken in the direction of arrows3--3 of FIG. 2;

FIG. 4 is a partly broken-away top view of a portion of the machine ofFIG. 1;

FIG. 5 is a sectional, side elevational view of a portion of the machineof FIG. 1 showing the head assembly of the machine in its lowerposition;

FIG. 6 is an enlarged top view of a portion of the assay tray shown inFIG. 1;

FIG. 7 is a sectional view taken in the direction of arrows 7--7 of FIG.6;

FIG. 8 is a sectional view taken in the direction of arrows 8--8 of FIG.7;

FIG. 9 is a sectional, partly schematic view of a portion of the machineof FIG. 1 showing the operation of the liquid withdrawal mechanism ofthe machine;

FIG. 10 is an enlarged top view of a portion of a second embodiment ofthe assay tray;

FIG. 11 is a sectional view taken in the direction of arrows 10--10 ofFIG. 10;

FIG. 12 is a sectional view taken in the direction of arrows 12--12 ofFIG. 11;

FIG. 13 is a sectional, partly schematic view corresponding to FIG. 9showing the operation of the machine to withdraw liquid from the assaytray of FIG. 10.

FIG. 14 is an enlarged top view of a portion of a third embodiment ofthe assay tray;

FIG. 14a is a sectional view taken in the direction of arrows 14--14 ofFIG. 14;

FIG. 15 is an enlarged top view of a portion of a fourth embodiment ofthe assay tray;

FIG. 15a is a sectional view taken in the direction of arrows 15--15 ofFIG. 15;

FIG. 16 is an enlarged top view of a portion of a fifth embodiment ofthe assay tray;

FIG. 16a is a sectional view taken in the direction of arrows 16--16 ofFIG. 16;

FIG. 17 is an enlarged top view of a portion of a sixth embodiment ofthe assay tray; and

FIG. 17a is a sectional view taken in the direction of arrows 17--17 ofFIG. 17.

MODES FOR CARRYING OUT THE INVENTION

The tray and machine are especially useful for conventional solid phaseimmunoassays. It will be appreciated, however, that the invention may beused for assays or heterogeneous reactions that involve physical and/orchemical interactions or reactions between substances other thanimmunochemicals. The particular trays and machine shown in the drawingsare designed to carry out enzyme immunoassays (EIAs), radioimmunoassays(RIAs), chemiluminescent immunoassays (CIAs) or fluorescent immunoassays(FIAs) in which the solid phase is a bead of an immunosorbent material.

The machine, which is best illustrated in FIGS. 1, 2, 4 and 5, is amodification of the machine that is described in copending, commonlyowned U.S. application SER. No. 06/489,866, filed May 5, 1983. Referringto those Figures, the present machine includes two main movable parts: ahorizontally translatable table 15 and a vertically translatable headassembly 16. The table 15 is mounted on hardened guide rods 17 by meansof slide bearings 18. The table is translated horizontally by action ofa stepper motor 19 through a pinion gear 22 connected to the motor and arack 23 mounted on the underside of the table. Head assembly 16 ismounted for vertical movement on guide rods 24 by means of slidebearings 25. Translation of the head assembly is provided by a steppermotor 21 via a similar pinion gear-rack arrangement or lead screwmechanism (not shown).

Head assembly 16 supports three main subassemblies: a pipette andplunger subassembly 26; a dispensing manifold subassembly 27; and anaspirating manifold subassembly 28. These three subassemblies are theparts of the machine that introduce liquids into the assay tray wellsand withdraw liquids therefrom. The pipette subassembly is an optionalfeature of this machine and is useful when it is necessary or desirableto be able to transfer precise amounts of liquid reagents to and fromthe tray. The details of the structure and function of the pipettesubassembly are described in said copending application Ser. No.06/489,866, which disclosure is incorporated herein by reference. Inview of this, only a brief description of this subassembly follows.

The pipette subassembly includes a series of pipettes 29 (FIG. 2) thatare arranged in a row transverse to the axis of translation of table 15.The pipettes are removably attached to the head assembly by means of amounting block 32. A plunger mechanism 33 is mounted on the headassembly for vertical movement relative to the pipettes. The plungermechanism includes a series of plunger rods 34, one being disposedrespectively within each pipette. All of the rods are mounted on acommon actuator bar 35 for concurrent vertical movement. The bar 35 istranslated along guide rods 36 by means of a stepper motor 37 and a leadscrew drive mechanism 38. Translation of the plunger rods relative tothe pipettes changes the internal air volumes of the pipettes, causing asimilar liquid volume to be aspirated into or expelled from the pipettetips 45.

The table 15 includes two work stations 39 and 42 for respectivelyaccommodating two trays 43, 44. Tray 43 is a conventional titer traythat includes a matrix arrangement of wells 41 for housing the liquid(s)to be taken up by the pipette tips. Instead of a matrix of wells, tray43 may be provided with one or more troughs that provide common liquidreservoirs from which liquid may be taken up. Tray 44 at the rear workstation 42 can be a tip tray that contains a similar arrangement ofreceptacles that accommodate disposable pipette tips 45. The tips 45 areinserted onto and engage the respective ends of the pipettes 29 when thehead assembly 16 is lowered by the stepper motor 21 after the table 15has brought one row of tips 45 into registry with the pipettes.

Once the tips are in place on the pipette ends, the table is translatedsuch that the pipettes are above and in registry with a row of wells intray 43 that contains the liquid to be taken up into the tips. Insteadof wells, tray 43 may have a common trough-like receptacle into whichthe tips are lowered. The tips are then lowered into the wells bytranslation of the head assembly and the tips are actuated by theplunger mechanism to withdraw a predetermined volume of liquid from thewells. The tips are then withdrawn from the wells by upward translationof the head assembly. As shown schematically in FIG. 5, the table isthen translated to bring assay tray 46 positioned at work station 47 ofthe table below and in registry with the tips. The tips are lowered bydownward translation of the head assembly into the wells 50 of tray andthe fluid is ejected by actuation of the plunger mechanism of thepipette subassembly. If desired, the pipette operation may be repeatedto extract more or other liquids from the titer tray and deposit it/themin the assay tray wells, with or without replacement of the tips, asdesired.

Removal of the tips 45 from the pipettes is accomplished with a tipejector means. The tip ejector means includes a stripper plate 48 thatis best illustrated in FIG. 2. The plate has openings that accommodatethe pipettes. The plate 48 is connected to and supported by a pair ofvertically translatable rods 49 mounted on the head assembly 16. Theserods are translated by means of a pair of solenoids 52 mounted on thehead assembly. When the solenoids 52 are deactuated, the ejector plate48 is maintained in its upper position. Actuation of the solenoids movesthe plate vertically downward, to push the tips 45 down and release themfrom their frictional engagement with the ends of the pipettes 29.

The dispensing manifold subassembly 27 is used to introduce liquids intothe wells 50 of the assay plate 46 except in instances where it isnecessary or desirable to introduce the liquid via pipette tip becauseof the availability of the liquid reagent or the need for precise volumecontrol. It is best shown in FIGS. 1, 2, and 3. It comprises a manifoldblock 53 that is mounted on the front of the head assembly such that itextends outwardly therefrom parallel to and above table 15. The block 53contains a plurality of horizontally spaced, parallel chambers 54 forreceiving the various liquids that are introduced into the wellsaccording to the protocol of the assay being conducted. The spacingbetween the vertical center lines of the chambers is the same as thespacing between the center lines of the rows of wells in the assay tray.Referring to FIG. 3, each chamber has a single inlet in its upper wallfitted with a tubular sleeve 55 and a plurality of equispaced outlets(six in the depicted manifold) each fitted with a liquid dispensing tube56 that extends vertically through the bottom wall of the block into achamber 54. Pressure tight conduits 57 are pressure fit over the outerend of each sleeve 55 and extend therefrom to the exhaust ports ofperistaltic (or similar fixed displacement pump) pumps 58. The inletports of the pumps are connected via another set of conduits 59 (FIG. 1)to containers 62 that hold the liquids that are used in the assay.

If expensive reagents are involved it will be desirable to use alternateassemblies of liquid containers and pumps that minimize dead volume.Such assemblies would involve a minimum of fluid passageways between theliquid reservoir and manifold. For instance self-contained syringe pumpunits mounted directly on the head assembly and stepper motors to drivethe syringe plungers might be employed. Accordingly, as used in theclaims the term "pump means" is intended to denote any device ormechanism that exerts the pressure (or suction in the case ofaspiration) required to effect the desired liquid transfer.

Aspirating manifold subassembly 28 is the part of the machine that isused to withdraw liquid from the wells of the assay tray as required bythe assay protocol. It includes an aspirating manifold block 63 mountedon the front of actuator bar 35 by means of arms 64 such that it isnormally located outwardly and above the leading end of the dispensingmanifold block 53. The aspirating manifold block contains a singlechamber 65 that has a plurality of equispaced inlets each fitted with aliquid aspirating tube 66. These tubes are longer than the dispensingtubes 56 so that when the dispensing and aspirating manifold blocks arein their uppermost positions the distal ends of tubes 56 and 66 lie in ahorizontal plane (FIGS. 9 and 13). The spacing between the tubes 66 isthe same as the spacing between the center lines of the columns of wellsin tray 46. The spacing between tubes 66 and the first row of dispensingtubes 56 along the axis of translation of table 15 is less than thediameter of the assay tray wells so that they may be positionedsimultaneously in the same row of wells 50. Chamber 65 has a singleoutlet fitted with a sleeve 67. A pressure tight conduit 68 is fit overthe outer end of sleeve 67 and extends to the inlet port of aperistaltic (or other fixed displacement pump) pump 69. The outlet portof pump 69 is connected via another conduit 72 to a receptacle 73 forreceiving liquids withdrawn from the assay tray wells.

FIGS. 6-17a illustrate the structure of the assay tray wells and theirrelationship to the liquid dispensing and aspirating tubes. The wellsshown in FIGS. 6-8 are cylindrical depressions, each of which has thesame structure. Each well has a flat, sloping bottom wall 74 that has adiametrical slot 75 in it. The slope of wall 74 and slot 75 both serveto cause the solid phase used in the assay, in this case spheres or"beads" 76, that are placed in the wells to assume by gravity uniformand relatively fixed positions. In this regard, the width of the slot 75is less than the diameter of the bead so that latter cannot slip downinto the slot. As shown the bead always assumes a resting positionseated in the slot at the low end of the sloping bottom wall. The bottomof the slot includes a section 77 that is parallel to the bottom wall ofthe tray and spaced horizontally from the low end of the bottom wall ofthe well and an upwardly sloping section 78 that is generally beneaththe position normally occupied by the bead. Slot 75 has severalfunctions. First, it provides a seat for the bead. Second, it provides adrain or sump for liquid that may be accessed vertically by theaspirating tubes 66 without disturbing the bead. Third, section 77provides an optically flat surface through which light may betransmitted in a vertical path that does not intersect the bead in orderto carry out optical measurements on the liquid contents of the well. Itwill be appreciated that the portion of section 77 that provides anoptical surface need not be flat but may define a lens or filter forfocussing or otherwise altering the light passing through it.

The well 79 depicted in FIGS. 10-13 differs structurally from the well50 of FIGS. 6-9, but functions in the same manner. This well consists ofthree sections: (a) an upper main well section 82, (b) a centrallylocated subwell 83 that provides a receptacle for the bead, and (c) asump section 84. Each of the three sections opens into the other. Uppersection 82 forms the mouth of the well and is generally frustoconical inshape, diverging outwardly directly from the interface with and openingof the subwell. Its shape causes the bead to drop into the subwell 83when the bead is placed in the well. The frustoconical shape of the mainsection also facilitates the flow of liquid through the subwell duringwashing steps and provides a sufficiently large liquid surface to permitoverhead fluorescent reading of the liquid contents of the well. In thisregard, the minimum diameter for FIA reading is approximately 8 mm. Thediameter of the main well section at its mouth will usually rangebetween 15 and 20 mm. The subwell is generally cyclindrical in shape.Its diameter is less than the average diameter of section 82 but greaterthan the diameter of the bead. Standard bead sizes are 4.4, 6.4 and 8mm. Accordingly the mean diameter of the subwell normally will rangebetween 6 and 10 mm depending on the size bead it is intended to hold.Preferred sizes are: 10 mm diameter for 8 mm bead; 8 mm for 6.4 mm beadand 6 mm for 4.4 mm bead. It has a concave shaped bottom wall. Sumpsection 84 is in the form of an elongated slot whose width and thicknessare less than the diameter of the bead so that the bead cannot fit intoit. It is at least as deep as the subwell 83 so that liquid can freelydrain into it from the subwell. The bottom wall of the sump is flat andparallel to the bottom wall of the tray. The configuration of the well79 serves the same function as that of well 50, with an additionaladvantage. The frustoconical shape of main section 82 and the beadreceptacle section combine to serve as a bead positioning means. Sumpsection 84 provides a fluid drainage area that may be accessedvertically without danger of disturbing the bead. The bottom wall of thesump section provides an optically flat surface for transmitting lightthrough the fluid contents of the well without intersecting the bead.The configuration and size of the subwell permit the bead to be immersedwith a relatively small volume of liquid. In this regard the volume ofthe subwell will normally be 30% or less of the volume of the entirewell, usually 10% to 25% of the entire well. In general, the minimumvolume of liquid to immerse the above described standard beads in thesubwell will depend upon the size of the bead and diameter of thesubwell. In the case of the above standard sized beads and using asubwell diameter about 1-2 mm larger than the bead diameter, the minimumvolume will be about 200 to 500 μl. This ability to immerse the solidphase in a minimum volume of liquid is important when the assay protocolcalls for use of expensive reagents. At the same time the upper liquidreservoir section allows the use of larger volumes of liquid such as aretypically used in washing the solid phase.

FIGS. 14 and 14a show a well 93 that has a pair of axial spaced ribs orpillars 94 having sloping flat top surfaces 95. The ribs define a rampthat causes the bead to drop into a bead receptacle section 96 of thewell. The space 97 between the ribs is less than the bead diameter anddefines a sump area that may be accessed vertically without disturbingthe bead.

FIGS. 15 and 15a and 16 and 16a depict trays in which the solid phasepositioning means is a plurality of spaced vertical posts positionedstrategically in the wells. The well 98 of FIGS. 15 and 15a has threeaxially elongated posts 99 with sloping top walls 102. The posts arealigned chordally and the distances between the posts and between theposts and the near portion of the cylindrical wall of the well issubstantially less than the diameter of the bead. When a bead is droppedinto the well this spacing and the sloping top walls 102 cause the beadto be positioned eccentrically in the well. The space between the postsand the near portion of the cylindrical well wall defines the sump areaof the well. FIGS. 16 and 16a show a well 103 that has four verticalposts 104. The posts are positioned in the four quadrants of the welland have top surfaces 105 that slope downwardly toward the center of thewell. This arrangement of wells causes the bead to be positinedcentrally in the well. Sump areas are located between the posts and thecylindrical wall of the well.

FIGS. 17 and 17a depict a well configuration that is similarconceptually to the well of FIGS. 16 and 16a. The well 106 of FIGS. 17and 17a has four radial ramps or fins 107 instead of the four posts 104of well 103. The top walls of fins 107 slope downwardly toward thecenter of the well, again causing the bead to be positioned centrally.Sump areas are located between the fins. The bottom walls of each ofwells 93, 98, 103, and 106 are flat so as to provide a flat surfacethrough which a light beam may be passed vertically in a path that doesnot intersect the bead.

The operations of pumps 58, 69, stepper motors 19, 21, 37 and solenoids52 are controlled by a microprocessor 85. The microprocessor controlsthe sequence of operations of each of these elements and thus theinterrelated movements of (a) the table on which the assay tray is held(b) the head assembly and the various parts thereof that movevertically, and (c) actuation of the pumps to transfer liquids to andfrom the wells. When the machine includes means tospectrophotometrically read the liquid or solid phase in the well themicroprocessor may also be used to control the spectrophotometer, recordthe readings made by it, process those readings or correlate them toother data, and generate reports based thereon.

As indicated previously, the apparatus shown in the drawings is designedto carry out EIAs. In EIAs one is typically looking for the presence orquantity of a multivalent antigen or hapten in a body fluid.Alternatively, EIAs may be used to look for the presence of a particularimmunoglobulin in a body fluid which is indicative of a particularmedical condition. In the former an immunoglobulin that is specific for(binds selectively to) the antigen/hapten of interest is immobilized onthe beads. In the latter the antigen is immobilized on the bead surface.Various types of materials are used to make the beads depending upon thenature of mechanism by which the immunochemical (antibody orantigen/hapten) is affixed to the bead surface. In this regard the term"immobilization" is intended to denote the ability to hold theimmunochemical by chemical (e.g., covalent bonding, hydrogen bonding) orphysical mechanisms (e.g., adsorption, hydrophobic-hydrophilicaffinity). Immunosorbent materials such as crosslinked dextran, agarose,silicated glass beads, polyacrylamides, polystyrene, or latex that bindthe immunochemical by adsorption are commonly used. It is expected thatfor commercial distribution the immunochemical-bearing beads will beprepared beforehand, placed in the wells, the wells sealed such as witha strippable cover and the trays packaged for storage or shipment. Thewells of a given tray may contain the same immobilized immunochemical ordifferent immobilized immunochemicals if the tray is to be employed fordifferent assays. Color coding or labels may be used to designate thecontents of the wells and/or the assay for which they are intended.

An EIA for an antigen/hapten will typically involve the followingsequence of steps.

1. Add samples and control reagents to the tray wells (and beads if theyhave not been pre-loaded)

2. Dilute sample with buffer (this is optional and the buffer may beadded to the well before or after the sample is added)

3. Incubate well contents

4. Withdraw samples/controls from wells

5. Wash beads with buffer (typically repeated 2-5 times which involvesaddition and withdrawal of buffer)

6. Add enzyme-labeled antibody reagent that binds specifically to theantigen/hapten

7. Incubate

8. Withdraw enzyme-labeled antibody reagent

9. Wash beads with buffer (again, typically repeated)

10. Add substrate

11. Incubate

12. Add enzyme deactivator (optional

in many assays)

13. Read wells spectrophotometrically.

Enzyme activity is related to antigen concentration and is determined bymeasuring the optical properties of the liquid contents of the wells atthe conclusion of the assay (step 13 above) and comparing thoseproperties to standard curves.

The invention machine would perform these steps in the following manner.The assay tray 46 preloaded with immunosorbent beads bearing the cognateantibody to the antigen/hapten is placed on table 15 at work station 47.Buffer is placed in the container 62 that feeds the first of chambers 54(i.e., the one nearest the aspirating manifold subassembly 28).Enzyme-labeled antibody reagent, substrate, and enzyme deactivator are,respectively, placed in each of the other containers 62. Predeterminedvolumes of samples/controls may be loaded into a titer tray 43 at workstation 39 and transferred therefrom to the wells 50 of tray 46 usingthe pipette subassembly as described above. After the samples andcontrols have been loaded into the assay tray wells the table istranslated so that a row of loaded wells is below the row of dispensingtubes 56 that communicate with the dispensing manifold chamber that isinterconnected to the container 62 holding the buffer. The dispensingmanifold is then lowered such that the ends of those tubes are over arow of wells. The peristaltic pump 58 that serves the buffer line isthen actuated to pump buffer into the dispensing manifold and out of thedispensing tubes into the wells. The samples/controls in the row ofwells being worked on is thereby diluted to the desired concentration.The table is then moved to position the next row of wells beneath thebuffer dispensing tubes, and the sequence is repeated to dilutesample/control in that row. Thus the sample/control in all or some ofthe rows may be diluted.

After the buffer dilution step the tray is incubated under conditionsthat permit any antigen/hapten in the sample/control to bind to thebead. If desired the machine can be equipped with a heating plate (notshown) that underlies the table 15 so that station 47 can be positionedover the heating plate to control the incubation temperature. Also, thetable 15 may be translated to position the tray 46 beneath a shroud 86to lessen the likelihood of the contents of the tray being contaminated.The table might also be agitated to facilitate mixing of the wells'contents through use of stepper motor drive 19. At the completion of theincubation, the table is translated to move the assay tray into positionfor the sample/control to be removed from the wells and the beads to bewashed. In this operation the row of wells to be worked on is positionedbeneath the aspirating tubes 66 and the first row of dispensing tubes 56(which dispense buffer). (As indicated previously the spacing betweenthe row of aspirating tubes and the first row of dispensing tubes issuch that both rows of tubes may be received in the same row of wells).The aspirating manifold is translated downwardly to move the aspiratingtubes down into the sump area of the wells (as shown in FIGS. 9 and 13).The dispensing manifold is concurrently translated downwardly to movethe buffer dispensing tubes over the same wells. The peristaltic pump 69is then activated, causing the spent sample/control to be aspirated intothe aspirating manifold, and pumped therefrom into waste liquidreceptacle 73. The peristaltic pump in the buffer line is then activatedto pump buffer (wash liquid) into the wells, thereby washing the bead.Waste wash, buffer is withdrawn by reactivating pump 69. By alternatingactivation of the aspirating pump 69 and the buffer line pump as manywash cycles as desired may be made. The aspirating tubes are thenwithdrawn from the row of wells and the table is moved, if desired, tocarry out wash operations on succeeding rows of wells.

Upon completing the washing, the table is translated to position a rowof wells beneath the dispensing tubes 56 that communicate with thechamber of the dispensing manifold that is interconnected to thecontainer 62 that holds the enzyme-labeled antibody reagent and thedispensing manifold is translated downwardly to lower those tubes intothe wells. The pump in the enzyme-labeled antibody line is thenactivated to cause a predetermined volume of that reagent to bedeposited in the wells. The dispensing manifold is then raised. Thissequence is repeated to deposit labeled reagent in other rows on thetray. After the labeled reagent has been added to the wells, the tray isagain incubated. Following incubation spent labeled reagent is withdrawnand the beads are washed using the same procedure that was used towithdraw spent sample/control and wash the beads previously. Substrateis then added to them by translating the table, lowering the substratedispensing tubes into the wells and activating the pump in the substrateline. Following another incubation, enzyme deactivator is added to thewells by moving the wells into position beneath the deactivatordispensing tubes, lowering them, and activating the pump in thedeactivator line.

After deactivation the optical properties of the liquid contents of thewells are read to measure enzyme activity. This may be done by removingthe trays from the machine and reading the wells with standard EIAreading equipment. Alternatively, the reading step may be carried outwithout removing the trays as follows.

Referring to FIG. 2, the machine is equipped with spectrophotometricmeans for carrying out the reading step without removing the tray. Thismeans includes a light beam generator 87 that underlies the table 15that is capable of generating beams of light 88 and a spaced,photoelectric receiver 89 mounted on the underside of shroud 86 oppositegenerator 87. The table has an aperture 92 in it at station 47 such thatthe bottom wall of the tray beneath the wells 50 is exposed. Inoperation, the table is translated to position the optically flatsections of the wells' sumps between the generator and receiver, thegenerator is activiated to pass the beam upwardly through the liquidcontents of the well to the receiver. The light energy received by thereceiver may be recorded and analyzed by conventional means. Theoperation of the generator and receiver and the recording and analyzingof the energy received by the receiver may also be handled bymicroprocessor 85. Microprocessor 85 may be linked to data storage means(e.g., disk drives) and read out means (CRT displays, printers) to storethe data and display it or the reports generated using it.

Other modifications of the above described embodiments of the inventionthat are obvious to those of skill in the fields of mechanicalengineering, immunoassay devices, robotics, and related fields areintended to be within the scope of the following claims. Suchmodifications may include, without limitation, altering the machine sothat the head assembly is horizontally movable and the table is fixedhorizontally or both are moveable horizontally. Also the horizontalmovement may be rotational rather than linear (e.g., the trays could becarried on a carousel with wells arranged in radial or circumferentialrows). The machine might also be altered to carry out FIAsautomatically. In such an embodiment the assay reading means wouldcomprise a fluorometer which would read the fluorescence of the liquidcontents of the wells. The predetermined location of the bead in thewell would enable accurate and reproducible direction of the excitationenergy and reading of the resulting emission energy. Further, wellconfigurations that are conceptually similar to the embodimentsdescribed in the drawings are readily apparent, such as configurationsthat comprise a bead receptacle well and a separate horizontally spacedsump that is interconnected via one or more channels to permit fluid tomove freely between the bead well or wells that contain screens orprojections other than the illustrated posts, ramps and fins.

We claim:
 1. A method of performing an automated assay in a machinehaving a moveable tray support, a tray having a plurality of assay wellsarranged in an array of rows and columns with each assay well made oflight transmitting material and configured to cause a solid phase beadto assume a position in a known area of the well and configured to havea sump region having a flat portion where the bead can never come torest and from which sump region liquid may be withdrawn and configuredsuch that a light beam may be passed through said flat portion with apath that will never be obstructed by said bead, and having a pluralityof dispensing tubes arranged in a plurality of rows with each rowcoupled to its own disengaging pump and its own surface of liquid usedin said assay, and having a plurality of aspirating tubes arranged in arow and coupled to an aspirating pump said aspirating tubes and saiddispensing tubes being mounted on moveable platforms so as to beindividually, vertically moveable comprising the steps of:(1) placingthe samples to be assayed and the appropriate control reagents in saidassay wells of a tray; (2) placing a tray at a workstation on said tablewherein each assay well has placed therein an immunosorbent bead whichhas immobilized thereon either a specific immunoglobulin for theantigen/hapten of interest or an antigen for which the antibody ofinterest is specific; (3) moving said table to register each row ofwells in turn with the row of dispensing tubes coupled to a reservoircontaining buffer solution and moving said dispensing tubes down intoeach row of assay wells in turn and activating the corresponding pumponce for each row of wells for sufficient duration to pump an adequateamount of said buffer solution into all the wells; (4) incubating thecontents of said tray; (5) moving said table to register each row ofsump regions in each row of wells in turn with said row of aspiratingtubes and moving said aspirating tubes down into said sump regions andactivating said aspirating pump long enough for each row of wells toaspirate the samples and reagents out of each well; (6) moving the tableand platforms for said aspirating and dispensing tubes and activatingsaid dispensing pumps and said aspirating pump appropriately to performthe following actions;registering each row of wells in turn with the rowof dispensing tubes coupled to said buffer reservoir, filling each wellwith buffer solution, aspirating the buffer solution out of each saidwell, and repeating these 3 actions a sufficient number of times to washeach said well and each said bead; (7) moving said table and saidplatform for said dispensing tubes appropriately and activating theappropriate dispensing pump so as to fill each well with an enzymelabelled antibody reagent that binds specifically to the antigen/haptenbound to said bead; (8) incubating the contents of said wells; (9)moving said table and the platform for said aspirating tubesappropriately and activating the aspirating pump appropriately toaspirate the enzyme labelled antibody reagent out of each said well;(10) repeating the washing actions of step 6 above; (11) moving thetable and platform for said dispensing tubes appropriately andactivating the appropriate dispensing motor to fill all the wells with asubstrate liquid; (12) incubating the contents of the wells; and (13)passing a beam of light from a spectrophotometer through said flatportion of said sump region and the liquid of each well and measuringthe level of light absorption.
 2. A method of performing an automatedimmunoassay using a machine having a moveable table and a tray having aplurality of assay wells therein where each well contains animmunosorbent bead having an antibody or antigen bound thereto andmaintains the immunoabsorbent bead out of alignment with an opticalanalysis path, the machine further having a plurality of rows ofdispensing tubes mounted on a platform which may be moved so as to movesaid dispensing tubes into and out of said wells, each row of saiddispensing tubes being coupled through a peristaltic dispensing pump toa separate reservoir for storing various liquids, and having a row ofaspirating tubes mounted adjacent to one row of said dispensing tubesclose enough that said dispensing tubes and said aspirating tubes mayboth be lowered into the same row of wells, said row of aspirating tubesbeing coupled to an aspiration pump and waste receptacle and mounted ona separately moving platform which enables the aspirating tubes to belowered into and raised out of said wells, and having a heating elementunder said table and a shroud over said table above said heating elementcomprising the steps of:(1) filling the reservoirs for four rows ofdispensing tubes with, respectively, buffer, enzyme-labelled antibodyreagent, substrate and enzyme deactivator appropriate for the automatedassay being performed where the buffer is loaded into the reservoircoupled to the row of dispensing tubes mounted adjacent to said row ofaspirating tubes; (2) loading each of the assay wells with sample liquidfrom a donor and with control reagent; (3) moving the table to registerone row of wells below the row of dispensing tubes coupled to the buffersolution reservoir and pumping buffer into the wells so registered; (4)repeating step (3) for all the other sample containing rows of wells;(5) moving the table over the heating element and under said shroud andcontrolling the heating element so as to control the temperature of theliquid in said wells for an appropriate incubation time; (6) moving thetable so that each row of sample containing wells is, in turn,registered under said row of aspirating tubes and lowering saidaspirating tubes and the adjacent row of dispensing tubes into each rowof wells so registered and activating the aspiration pump to pump outliquid from each said well and activating said dispensing pump to pumpsaid buffer into said row of wells and repeating this aspirating anddispensing of buffer cycle a sufficient number of times to wash eachsaid bead and each said well in each said row of wells; (7) registeringeach row of wells below the row of dispensing tubes coupled to saidreservoir containing enzyme labelled antibody and pumping same into eachwell; (8) moving said table to place said tray over said heating elementand under said shroud and controlling said heating element to incubatethe contents of the wells; (9) registering each row of wells under saidrow of aspirating tubes and lowering said aspirating tubes into each rowof wells in turn and aspirating out the liquid in each said well; (10)washing each said well using the procedure of step (6) above; (11)registering each row of wells in turn beneath the row of dispensingtubes coupled to the reservoir containing substrate and pumpingsubstrate into each well; (12) moving the table to place the tray undersaid shroud and over said heating element and controlling said heatingelement to incubate the contents of said wells; (13) registering eachrow of wells under the row of dispensing tubes coupled to the reservoircontaining enzyme deactivator and pumping same into each well; and (14)maintaining said bead out of alignment with said optical path andpassing a beam of light through the liquid in each well along saidoptical path and reading the amount of light transmission through saidliquid.
 3. The method of claim 2 where said machine includes aspectrophotometer having a light source under said table at apredetermined position to generate beams of light up through said tabletoward a row of photodetectors mounted on the underside of said shroudand wherein each said well is configured so as to positively control thepositive of said bead to a predetermined location under the influence ofgravity and wherein step 14 comprises the steps of:moving each row ofwells in turn into registry with said light beams such that light beamspass through the liquid of said wells without being obstructed by saidbead; and reading the amount of light absorption by the liquid in eachwell.
 4. An apparatus for automatically performing immunoassays using aspectrophotometer comprising:a spectrophotometer means for projecting abeam of light and for measuring the light intensity in said beam oflight at a position removed from the position of the source of said beamof light; a tray having a plurality of assay wells arranged in rows,each well for containing liquids needed in said assay and a solid phasehaving immobilized thereon a specific immunoglobulin for theantigen/hapten of interest or an antigen for which the antibody ofinterest is specific; means in each assay well for preventing said solidphase from ever residing in a place in said well which would block thepath of said light beam used to measure the results of said assay; ahead assembly translatable along a vertical axis; at least one liquidaspirating manifold mounted on said head assembly having an outlet and aplurality of spaced inlets each fitted with a plurality of verticallydepending liquid aspirating tubes arranged in a row with spacing tomatch the spacing of the rows of said assay wells; means mounting saidliquid aspirating manifold to said head assembly for moving said liquidaspirating manifold vertically even when said head assembly is notmoving; sump means in each said well for providing a location from whichall the liquid in each said well may be removed by an aspirating tube atany time without interference between said solid phase and saidaspirating tube; a plurality of liquid dispensing manifolds mounted onsaid head assembly and having a plurality of inlets, each adapted to beconnected to a source of a different liquid to be used in said assay,each said liquid dispensing manifold having a plurality of spacedoutlets each fitted with a vertically depending liquid dispensing tube,the spacing between said dispensing tubes being in correspondence withthe spacing between said wells of said assay tray; a plurality ofdispensing pump means each coupling one of said liquid sources to one ofsaid liquid dispensing manifolds for pumping liquids from said sourceout the corresponding ones of said dispensing tubes; an aspirating pumpmeans coupling said aspirating manifold to waste for pumping liquidsfrom the sump regions of any assay wells that said aspirating tubes arein; means for moving said head assembly along its vertical axis; a tablemounted beneath said head assembly and adapted to carry said tray, saidtable being movable horizontally relative to said head assembly; meansfor moving said table horizontally so as to position any said row ofsaid wells in registry with any said row of dispensing tubes or withsaid row of aspirating tubes; incubation means located adjacent to saidtable for controlling the temperature of fluid in said wells; means forcontrolling each of said moving means for said head assembly and saidtable, said dispensing pump means, said aspirating pump means, saidspectrophotometer means and said incubation means such thatpredetermined volumes of predetermined liquids are added to and removedfrom said wells in a predetermined sequence and such that the liquids insaid wells are subjected to incubation times at controlled temperaturesfor intervals at predetermined times in said sequence and for causingsaid table to move said table such that each well is moved into the pathof a light beam from said spectrophotometer at a predetermined time insaid sequence such that said light beam passes through the liquid insaid well without being blocked by said solid phase and the lightabsorption by said liquid is measured.